Structure characterization and immunoactivity on dendritic cells of two neutral polysaccharides from Dictyophora rubrovalvata

Dictyophora rubrovalvata is a valuable fungus homologous to food and medicine, and its polysaccharide have been gaining increasing attention because of its plentiful activity. However, the structure and activity of its homogeneous polysaccharide have not been studied enough. In this study, two polysaccharides DRP-I and DRP-II were purified from D. rubrovalvata. Their structures were characterized by chemical composition, monosaccharide composition analysis, methylation analysis and nuclear magnetic resonance spectroscopy. The results showed that DRP-I and DRP-II were neutral heteropolysaccharides with molecular weights of 5.79 × 103 and 1.25 × 104 Da, respectively, which were composed of mannose, galactose, glucose, xylose and fucose. The main chains were → 6)-α-D-Galp-(1 → 6)-α-D-Galp-(2,1 → 6)-α-D-Manp-(2,1 → 6)-α-D-Galp-(1, and branch chains were β-D-Xylp-(1 → 3)-α-L-Fucp-(1 → 4)-α-D-Manp-(1 → and α-D-Galp-(1 → 3)-α-D-Galp-(1 → . The in vitro immunoactivity assays on dendritic cells showed that DRP-I and DRP-II could up-regulate the expression of IL-10 and IL-6 and inhibit the expression of TNF-α in a concentration-dependent manner. This research indicated that DRP-I and DRP-II possessed immunoactivity by balancing the excessive inflammation, and molecular weight is an important factor affecting immunoactivity. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-024-00476-6.


Graphical Abstract 1 Introduction
Edible mushroom polysaccharides are important natural active macromolecules, which have attracted extensive attention due to their unique structures and abundant biological activities [1].It has been reported that most of polysaccharides in edible mushroom are composed by D-glucan, and there are also heteropolysaccharides such as mannose, arabinose and galactose [2,3].And exhibiting a variety of biological activities such as immunomodulatory, anti-inflammatory, hypoglycemic, and anti-tumor [4][5][6][7].Immunoactivity is one of the most important biological activities of edible mushroom polysaccharides, they can exert immunoactivities by influencing the expression of cytokines, activating MAPK and other signaling pathways, and indirectly acting on intestinal flora [8].Structure-activity relationship studies have shown that the activity of polysaccharides is affected by polymerization degree (molecular weight), monosaccharide composition, type of glycosidic bond, branching degree and higher conformation [9].
Dictyophora rubrovalvata is one of the traditional edible and medicinal mushrooms belonging to Dictyophora genus, Phallaceae, Basidiomycota, which is identified by professor Zang Mu and cultivated wildly in China nowadays [10].Its mature fruiting body includes thallus (consists of stipe and indusium), pileus, and volva [11].Previous researches showed that polysaccharides were the mainly active components of D. rubrovalvata, and they exhibited satisfactory immunoenhancing, anti-tumor, antihyperlipidemic and antioxidant effects [12][13][14][15].These indicated that D. rubrovalvata was an important source of active polysaccharide.However, the structures and activities of homogeneous polysaccharides still need further study.
In this study, two neutral polysaccharides (DRP-I and DRP-II) have been purified from thallus and volva of D. rubrovalvata.Meanwhile, their structures were characterized by molecular weight, chemical compositions, monosaccharide compositions, methylation analysis and        UV, IR and NMR spectroscopy.In addition, the in vitro immunoactivity was evaluated using dendritic cells, including cell proliferation capacity and cytokine (IL-10, IL-6 and TNF-α) secretion capacity.

Isolation and purification
Through the hot water extraction, alcohol precipitation and purification by ion exchange column and gel column, two neutral polysaccharides named DRP-I and DRP-II were obtained, respectively.As shown in Fig. 1A, they exhibited single and symmetrical sample peaks analyzed by high-performance gel permeation chromatography (HPGPC), which indicated that they were homogeneous polysaccharides.

Molecular weight and chemical compositions determination
The molecular weights of DRP-I and DRP-II were determined using HPGPC.The contents of total sugar and protein of them were determined by the phenol-sulfuric acid method and Bicinchoninic-acid method.As shown in Table 1, the molecular weight of DRP-I and DRP-II were 5.79 × 10 3 Da and 1.25 × 10 4 Da, and the total sugar contents were 92.78% and 88.43%, respectively.The results of UV spectrum (Fig. 1B) and protein detection assay (Table 1) pointed out DRP-I and DRP-II contained almost no other impurities.

Infrared spectral analysis
The FT-IR spectrums of DRP-I and DRP-II were shown in Fig. 2B, they both presented the characteristic peaks of polysaccharide.The strong and wide absorptions at approximately 3300-3400 cm −1 attributed to the O-H stretching vibration, the peaks at about 2925 cm −1 were the stretching vibration of C-H from CH, CH 2 and CH 3 , the absorption peaks at about 1640 cm −1 were caused by water, and the C-O stretching vibration and O-H bending vibration appeared in approximately 1460-1200 cm −1 [16].In addition, the three absorption peaks at 1200-1000 cm −1 are C-O-C stretching vibration of pyranoid sugar ring [17].

Methylation analysis
DRP-I and DRP-II were methylated, acetylated, GC-MS analyzed and compared the m/z results of PMAAs with the Complex Carbohydrate Research Center database (https:// www.ccrc.uga.edu) to obtain linking information.The results were presented in Table 2, Fig. S1, and Fig. S2, indicating that DRP-I and DRP-II were complex polysaccharides with 10 linkage fragments.

NMR analysis
NMR analysis was used to further elucidate the structures of DRP-I and DRP-II.In generally, the polysaccharide's chemical shifts of anomeric hydrogen and anomeric hydrogen of the α-configuration are δ H 5.1-5.8ppm and δ C 98-103 ppm, while the corresponding chemical shifts of the β-configuration are δ H 4.3-4.8ppm and δ C 103-106 ppm [18].The 1 H, 13 C and HSQC spectra of DRP-I showed that there were 11 kinds of anomeric hydrogen Therefore, combined with the results of monosaccharide composition analysis, methylation analysis and NMR analysis, DRP-I was the branched neutral heteropolysaccharide with the proposed repeated unit derived in Fig. 3G.Moreover, DRP-II had similar monosaccharide composition, linkage fragments, and NMR spectrums with DRP-I, suggesting the similarity of their structures (Fig. S3 and S4).Comparing with the ultrasonic assisted water-extracted polysaccharides from pileus of D. rubrovolvata previously, DRP-I and DRP-II have different molecular weight, monosaccharide composition and linkages, suggesting that the structural features of polysaccharides may depend on extraction part and method [15].

SEM analysis
Scanning Electron Microscope (SEM) detection results of DRP-I and DRP-II (Fig. 4A) showed that the microscopic apparent form of DRP-I is lamellar with pores, while DRP-II is rod-like, spheroidal and lamellar form.DRP-I exhibits a more orderly apparent morphology.

Congo red analysis
When the Congo red and triple-helix conformation polysaccharide form the complex at a range of low base concentration, its maximum absorption wavelength (λmax) moves towards a long wavelength, so that the triple-helix conformation of polysaccharide can be identified by Congo red experiment [28].The λ max of congo red and DRP-I ~ DRP-II + congo red at different NaOH concentrations were shown in Fig. 4B.At 0.00-0.20 M NaOH, λmax decreased first and then tended to be stable.These results indicated that DRP-I and DRP-II did not have triple-helix conformation.

Immunoactivity on dendritic cells
The immunoactivities of DRP-I and DRP-II were evaluated using dendritic cells.The effect on proliferation was determined by MTS method, as shown in Fig. 5A and B, DRP-I and DRP-II showed no cytotoxicity at concentrations ranging of 100-400 μg/mL.The effects of DRP-I and DRP-II on cytokines (Fig. 5C, D and E) showed that they could promote the secretion of the anti-inflammatory cytokine IL-10 and the pleiotropic cytokine IL-6 and inhibit the secretion of the pro-inflammatory cytokine TNF-α with dose-dependent manner, and the immunoactivity of DRP-I was more significant.It might be suggested that DRP-I and DRP-II balancing the excessive inflammation to perform immunoactivity [29].Based on the receptor-active center theory, polysaccharides with lower polymerization degree are more likely to bind to the active receptor center, which may be the reason for the more significant activity of DRP-I [30].

Discussion and conclusion
In this work, two polysaccharides named as DRP-I and DRP-II were purified from D. rubrovalvata, with molecular weights of 5.79 × 10 3 and 1.25 × 10 4 Da, respectively.They were composed of mannose, glucose, galactose, xylose and fucose.And the main chains were . DRP-I and DRP-II showed immunoactivity by up-regulating the expression of IL-10 and IL-6 and inhibiting the expression of TNF-α on dendritic cells, in which DRP-I had more significant immunoactivity.In summary, DRP-I and DRP-II are potential natural immunomodulators, and the molecular weight of polysaccharides is closely related to immunomodulatory activity, which lays a foundation for the development of immunomodulatory polysaccharides and the study of their structure-activity relationship.

Chemicals and reagents
The fresh D. rubrovalvata was obtained from Songming, Kunming, China in July 2022.The samples were identified by Zhao Qi, a senior engineer at the Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and purification
The fresh fruiting bodies of D. rubrovalvata were divided into volva, thallus and pileus.They were soaked with 95% ethanol to remove pigments and other alcohol-soluble compounds (3 times, each time for 12 h), and ventilated at room temperature to dry, respectively.The dried thallus of D. rubrovalvata were crushed and extracted with hot water (w: v = 1:20, 3 times, each time for 2 h).The water extract was collected by centrifuging, and then concentrated.The protein of water extract was removed by the Sevage method (chloromethane: n-butanol = 4:1, v/v).Subsequently, the water extract was mixed with anhydrous ethanol for the final ethanol concentration to reach 60% (v/v) to precipitate polysaccharides.The precipitates were re-dissolved in hot distilled water and removed the residual ethanol.At last, the crude thallus and volva polysaccharides of D. rubrovalvata (DRTP60 and DRVP60) were obtained by lyophilization.
The crude polysaccharides (DRTP60 and DRVP60, 10 mg/mL) were subjected on DEAE-52, which were eluted with distilled water, 0.1 M NaCl, 0.3 M NaCl and 0.5 M NaCl solution at a flow rate of 2.0 mL/min.And the concentrated eluents were further purified on the Sephacryl S-200 and Sephacryl S-300 gel permeation columns.The columns were eluted with distilled water at a flow rate of 0.5 mL/min.The eluents were tested by by HPLC system (Agilent, USA) equipped with the evaporative light scattering detector (ELSD, Alltech, USA) and Shodex KS-804 column (7.8 mm × 300 mm) and the same fractions were combined.And then the purified and single fractions (DRP-I and DRP-II) were obtained by lyophilization.

Chemical compositions
The total sugar content was measured with phenol-sulfuric acid method [31].The protein content was determined by Bicinchoninic-acid method [32].And the UV spectrophotometer was used to detect the presence of proteins and nucleic acids that have ultraviolet absorption by scanning in the range of 190-600 nm.

Molecular weight and homogeneity determination
The average molecular weight and homogeneity of DRP-I and DRP-II were determined by HPLC-ELSD system equipped with Shodex KS-804 column (7.8 mm × 300 mm).The standard dextrans with different molecular weights (1,5,12,410, 670 kDa) were used to establish a standard molecular weight curve.

Monosaccharide composition analysis
The analytical method of monosaccharide compositions of DRP-I and DRP-II referred to Dai et al. with Simple modification [33].2.0 mg of polysaccharides DRP-I and DRP-II were hydrolyzed with 4 mol/L TFA at 90 °C for 8 h, and the residual TFA was removed by adding methanol repeatedly and drying under pressure.The hydrolyzed sample and monosaccharide standard (arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, xylose and ribose) were derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP).They were prepared into the solutions of 1 mg/mL and mixed with 50 μL of NaOH (0.6 M) and 100 μL of PMP (0.5 M), respectively.The reaction was performed at 70 ℃ for 100 min.After the reaction and cooling to room temperature, the mixtures were added 100 μL of HCl (0.3 M), 1 mL distilled water and 1 mL chloroform.The supernatant was extracted after chloroform extraction (3 times) and filtered for HPLC analysis.

Infrared (IR) spectral analysis
Approximately 1 mg of dried polysaccharides of DRP-I and DRP-II evenly mixed with KBr powder, tableted.And then they were analyzed with FT-IR spectroscopy (Bruker, Germany) in the range of wave length 4000-500 cm −1 .

Methylation analysis
The analytical method of methylation of DRP-I and DRP-II referred to Ciucan, Kerek and Liang et al. with Simple modification [34,35].5.0 mg of polysaccharides DRP-I and DRP-II were dissolved with anhydrous DMSO completely, filled with N 2 gas, and then NaOH (20 mg) was added.After ultrasonic mixed for 20 min, the mixture was cooled and solidified.Then dropping CH 3 I (1.5 mL) slowly and ultrasonic reacted for 30 min.The methylation reactions were terminated by adding 1 mL distilled water.Repeat the above steps until the methylation was complete.The methylated polysaccharides were hydrolyzed with 4 mol/L TFA at 110 °C for 4 h.Subsequently, NaBD 4 (10 mg/mL) was added to reduce samples, and acetic anhydride / anhydrous pyridine (v/v = 1:1) were added to acetylate (120 ℃, 120 min).The acetylation products were extracted by CH 2 Cl 2 for GC-MS analysis.

NMR analysis
The freeze-dried polysaccharides of DRP-I and DRP-II (10 mg) were dissolved in D 2 O (0.5 mL).Repeatedly freeze-dried to replace the H in the polysaccharides with D. The 1D and 2D NMR data were measured by Bruker Advance 800 MHz NMR spectrometer (Bruker, Germany).

SEM analysis
The freeze-dried polysaccharides of DRP-I and DRP-II attached on the conductive adhesive and gold-plated.And then the samples were observed their surface morphology at different magnifications by scanning electron microscope (Carl Zeiss, Germany).

Congo red analysis
Congo red assay was used to analyze whether the polysaccharide had a triple-helix conformation [36], and the steps were as follows: In the experimental group, polysaccharide solution (2 mg/mL) was mixed with Congo red solution (80 μM) in equal volume (50 μL), and then 100 μL NaOH solution with different concentrations was added respectively.The final concentrations of NaOH in the mixed solution were 0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.45 M, respectively.After incubating at 25 ℃ for 10 min, the UV absorption of the samples in the range of 400-700 nm was analyzed by microplate reader.The control group was deionized water mixed with Congo red solution in equal volume, and the specific steps were the same as above.

Proliferation assay
The proliferation of DRP-I and DRP-II were determined by MTS method on BMDCs [4].100 μL of BMDCs (1 × 10 5 cell/mL) were cultured in 96-well cell plates, after the cells were attached to the wall, the supernatant was discarded.The experimental group was added with DRP-I and DRP-II (100, 200 and 400 μg/mL, 200 μL), and the blank control group was added with RPMI-1640 base medium (200 μL).The culture was continued for different time (24 h and 48 h).Then the supernatant of cells in the 96-well plate was discarded, and MTS solution (20 μL) and RPMI-1640 base medium (100 μL) were added to each well, and cultured under the same conditions for 1 h.The absorbance of each well was measured at 490 nm by microplate reader.

Fig. 1
Fig. 1 High-performance gel permeation chromatography profiles of DRP-I and DRP-II (A).The UV spectra of DRP-I and DRP-II (B) molecular weight, chemical and monosaccharide compositions of DRP-I and DRP-II

Fig. 2
Fig. 2 The HPLC profiles of monosaccharide analysis of DRP-I and DRP-II (A).The FT-IR spectra of DRP-I and DRP-II (B)

Table
The

Table 2
The glycosidic linkages of DRP-I and DRP-II determined by methylation analysis

Table 3
The 1 H and13C chemical shifts of DRP-I